Analysis of biological samples

ABSTRACT

The invention provides a method of analysing a biological sample of interest by use of: i) a probe library which comprises cDNA (or a derivative thereof) representative of a pattern of multiple gene expression in the biological sample of interest; and ii) a plurality of individual reference samples (preferably provided as an array on a substrate) each of which is a library comprised of cDNA (or a derivative thereof) representative of a pattern of gene expression in reference biological samples from which the reference samples have been derived. The method is effected by treating individual reference samples with the probe library under hybridising conditions. The relative degree of hybridisation of the probe library to the reference samples is then determined, thereby providing an indication of the degree of similarity between gene expression in the biological sample of interest and gene expression in the individual reference biological samples. The greater the level of hybridisation between the probe library and reference samples the greater the degree of similarity in the patterns of gene expression in the samples from which they are derived.

The present invention relates to a method of analysing a biologicalsample to determine a pattern of gene expression therein.

Recent years have seen a growth in the realisation of the importance ofgene expression in the control of biological activities. It is knownthat expression of specific subsets of genes regulate tissue formationand organogenesis during development and also the properties of adulttissues. Patterns of gene expression influence not only the structureand composition of specific tissues, but also the tissues responses tovarious stimuli. These structures, composition and responses, and thepatterns of gene expression encoding them, are distinctive markers forindividual tissues.

At a more complex level the pattern of genes expressed by wholeorganisms may be characteristic of specific individuals and provide aninsight into their biological status. For instance, there is growingevidence that the pattern of genes expressed by an individual mayinfluence factors such as the individual's predisposition to particulardiseases or their responsiveness to certain therapeutic agents.

The current challenge to biologists is to learn how the products of thearound 40,000 identified human genes interact to produce the complexityexhibited by higher eukaryotes. To a large extent the biologicalcharacter of a cell can be inferred from the profile of genes itexpresses. Although an examination of mRNA or protein expressionpatterns alone does not directly address function, the knowledge of whenand where a gene is expressed can provide valuable insights as to thepotential role of a gene and has historically been instrumental in thediscovery of developmentally regulated genes. Recognition of the valueof the examination of expression patterns led to the development of aplethora of advanced mRNA profiling technologies such as cDNAmicroarrays (Duggan et al., 1999), SAGE (Velculescu et al., 1995), andcDNA display (Liang and Pardee, 1992) aimed at the simultaneousmeasurement of tens to several thousand genes in the target samples.Application of these profiling technologies to clinical diseases, suchas cancer has confirmed the utility of profiling and provided usefuldiagnostic and prognostic assays (Shipp et al., 2002; Staunton et al.,2001; van't Veer et al., 2002).

Despite the success of these approaches at the molecular level byidentifying patterns of expression exhibited generally by relativelyhomogeneous cellular samples the cellular complexity of highereukaryotes still presents a major obstacle to expression profiling.

Over the last 30 years a variety of molecular techniques have beendeveloped for the analysis of gene-expression. In general methodsfocussed either on the identification and characterisation of genes(either individual genes or networks of related genes) or thecharacterisation of the input tissue or cell based on a characteristicprofile of expressed genes. Although conventional nucleic acidhybridization techniques (such as northern and dot blots) have been usedfor many years to analyse a small number of genes and samples there havebeen a variety of advanced mRNA profiling technologies such as cDNAmicroarrays (Duggan et al., 1999), SAGE (Velculescu et al., 1995), andcDNA display (Liang and Pardee, 1992) which have been recently developedto allow the simultaneous measurement of tens to several thousand genesin the target samples. In order to take both full advantage of and toextend recent improvements in gene-expression analysis it is importantthat the final processed sample in the form of RNA or cDNA is compatiblewith a wide variety of expression profiling methods.

It is an object of the present invention to obviate or mitigate thedisadvantages associated with the prior art.

According to a first aspect of the present the present invention thereis provided a method of analysing a biological sample of interest,comprising:

-   -   (i) providing a probe library which comprises cDNA or a        derivative thereof representative of a pattern of multiple gene        expression in the biological sample of interest;    -   (ii) providing a plurality of individual reference samples each        being a library comprised of cDNA or a derivative thereof        representative of a pattern of gene expression in reference        biological samples from which the reference samples have been        derived;    -   (iii) treating individual reference samples with the probe        library under hybridising conditions; and    -   (iv) determining the relative degree of hybridisation of the        probe library to the reference samples, thereby providing an        indication of the degree of similarity between gene expression        in the biological sample of interest and gene expression in the        individual reference biological samples.

According to a second aspect of the invention there is provided acollection of individual reference samples each being a librarycomprised of cDNA or a derivative thereof representative of a pattern ofgene expression in reference biological samples from which the referencesamples have been derived.

The present invention makes it possible to obtain an indication of thedegree of similarity of the pattern of gene expression in a sample ofinterest with that in a number of reference samples. The greater thedegree of similarity of the pattern of gene expression in the sample ofinterest with a particular reference sample then the greater thesimilarity between these two samples. The ability to compare geneexpression in a sample of interest with that in a large number ofreference samples is, of course, particularly advantageous where thereference samples are of well characterised biological status sinceconclusions may then be drawn as to the biological status of the sampleof interest.

A library for use according to the method of the invention is acollection of individual sequences representative of gene expressionwithin the biological sample from which the library is derived. Thenumber of sequences in the collection is sufficient to providesignificant information about the biological activity or status of thebiological sample from which the library is derived. Thus, although abiological sample may express many thousands of genes a library may, forinstance, represent ten or more genes the expression of which arecharacteristic of the activity or status of the biological sample.Preferably the probe library may represent twenty or more genes theexpression of which are characteristic of the biological sample. Mostpreferably the probe library may represent fifty or more genes theexpression of which are characteristic of the activity or status ofbiological sample.

More particularly the method of the invention utilises a probe librarycomprising cDNA, or a derivative thereof, representative of the mRNAcontent of, and hence a pattern of gene expression in, the sample ofinterest. The individual reference samples are each libraries comprisedof cDNA molecules representative of gene expression in the biologicalsamples from which the reference samples are derived. The libraries may,for example, comprise total cDNA. Examples of cDNA derivatives that maybe employed in the method of the invention include sub-populations oftotal cDNA (e.g. obtained by complexity reduction techniques), andderivatives obtained by partial exonuclease digestion of the cDNA. Theterm cDNA derivative also includes RNA obtained from any form of DNAthat may be employed in the invention.

The comparison of the patterns of gene expression in the sample ofinterest and the reference samples may be effected by probing thereference samples with the probe library under conditions allowinghybridisation of molecules in the two samples that are complementary toone another. It is preferred that the probe library is labelled forpurposes of detecting hybridisation. If a particular gene is beingexpressed in both a sample of interest and a reference sample then theprobe representing that gene will hybridise to the corresponding cDNA inthe reference sample. This hybridisation may then be detected. Thegreater the level of hybridisation between the probe library andreference samples the greater the degree of similarity in the patternsof gene expression in the samples from which they are derived.

The ability to compare overall gene expression in a sample of interestwith that in a number of reference samples is particularly advantageouswhen the reference samples are of defined and well-characterisedbiological status since conclusions may then be drawn as to thebiological status of the sample of interest.

The method of the invention need only require a single round ofhybridisation to allow comparison between the pattern of expression of aplurality of genes in a sample of interest with the pattern ofexpression of the same genes in a number of reference samples. Thepattern of expression may potentially extend to the expression ofthousands of different genes. Since known techniques only analyse theexpression of either small numbers of genes or small numbers of samplessuch information could only be provided by the prior art on completingmultiple rounds of hybridisation. Thus the method of the inventionprovides advantages both in terms of a reduction in the time necessaryto perform such a comparison, and also in the reduced amount of reagentsrequired.

In contrast to existing methodologies (in which specific probes are usedto investigate the expression of specific genes) the method of theinvention is able to compare patterns of gene expression withoutrequiring any specific information as to the genes involved. Thus it isnot necessary to identify those genes that may be of interest beforecomparing patterns of gene expression between unknown and referencesamples. This provides a considerable advantage over the prior art inthat an investigator does not need to know what gene (or genes) areinvolved in, for example, a particular response to a therapeutic agentbefore he can establish whether a test subject is likely to respond in asimilar way to a previously characterised subject with a known response.

The reference samples may be derived from biological reference samplesrepresenting a number of different biological conditions or states.Alternatively the reference samples may be derived from biologicalreference samples representing a number of different examples of thesame biological condition or state. Individual reference samples may bederived from biological samples taken from one or more individual. Inthe instance that a reference sample is derived from a single individualthe reference sample may be derived from a biological samplerepresenting a single tissue, or from biological samples representing anumber of different tissues. In the case of reference samples derivedfrom biological samples taken from more than one individual thebiological samples may all represent one type of tissue, or mayrepresent a number of different tissue types. By including referencesamples which share a common biological phenotype yet have arrived atthat state via different routes the method of the invention is able todiscriminate between treatment and biological status.

Examples of materials that may be used as reference samples includesamples which are derived from patients with known clinical conditionsand/or with known clinical outcomes.

In one such example reference samples may be taken from a number ofpatients with different forms of a particular disease. In this instancethe sample of interest may be taken from an individual suspected ofhaving, or being predisposed to, the disease in question. By comparingthe pattern of gene expression in the sample of interest with thepatterns of gene expression in the reference samples it is possible toestablish which of the reference samples the sample of interest mostclosely resembles. This may then in turn provide an indication as to theparticular form of the disease in question that the individual testedhas or is predisposed to.

Alternatively the reference samples may be derived from patients withthe same disease, but having different (known) reactions to differenttherapeutic agents. In this case comparison of the sample of interestand the reference samples will establish which of the patients withknown treatment history the individual providing the sample of interestmost resembles. This knowledge can then be used in order to select thetreatment regime believed most likely to produce a beneficial outcomefor the individual in question.

In another alternative reference samples may be derived from the samepatient at different times, for instance before, during and aftertherapy. Comparison of such samples with a sample of interest taken froma patient with the same disease may be useful in assessing the progressof the patient of interest during treatment.

In a further alternative suitable reference samples may be collectedfrom experimental subjects, such as animals or cultured cells, that haveundergone procedures the effects of which are well studied.

For example reference samples may be collected from cells of a cell linethat have been exposed to different drugs that have known effects(either on the cell line or on organisms of interest). These samples maythen be probed using a probe library derived from cells of the same cellline that have been exposed to a putative drug which has an unknowneffect. By comparing gene expression patterns established in response tothe known and unknown drugs it is then possible to establish which ofthe known drugs the unknown drug most resembles. This will provide anindication that the effects of the unknown drug are likely to be similarto those of the known drug that it most closely resembles.

In a further example the sample of interest may be taken from tissues ofexperimental animals that have undergone treatments bringing aboutconditions that resemble those of a disease of interest. The pattern ofgene expression in these samples may then be compared with the patternof gene expression in reference samples taken from normal biologicalsamples or biological samples from individuals suffering from thedisease in question in order to investigate how changed gene expressioninfluences the particular disease. Suitable experimental animals may,for instance, include transgenic animals, such as animals in whichcertain genes have been up-regulated, down-regulated or deleted.

In another application of the method of the invention the sample ofinterest may be taken from a tissue that includes, or may be thought toinclude, a cell type of particular interest. Such cells may, forexample, be stem or progenitor cells. In this case tissues representingsuitable biological samples from which reference samples may be derivedwill include tissues known to contain the cell type of interest, ortissues known to contain specific forms of the cell type of interest.Comparison of the pattern of gene expression in the sample of interestwith the pattern of gene expression in the reference samples mayindicate that the sample of interest either does or does not contain thecell type of interest. If the sample of interest contains cells of thecell type of interest then the method of the invention may also provideinformation as to the number, form or status of these cells present inthe sample.

In the field of stem cell biology defining the specific gene expressionchanges in stem cells, their immediate daughter cells, cells committedto differentiation and fully differentiated cells under conditions thatalter self-renewal and differentiation represents a powerful means ofidentifying potential drug targets. For example, the discovery of agrowth factor or growth factor receptor specifically expressed in stemcells undergoing increased self-renewal would lead to the development ofpharmacological approaches designed to inhibit stem cell expansionduring cancer development or increase stem cell expansion followinginjury. Furthermore, identification of genes whose expression isspecifically linked to eventual stem cell self-renewal anddifferentiation will greatly facilitate the monitoring of stem cellbehaviour that is an essential component of pre-clinical drugevaluation.

Since the method of the invention compares the patterns of expression ofa number of genes within test and reference samples it is particularlywell suited to the study and comparison of biological activitiesassociated with stem cells that involve the interplay of a number ofdifferent genes, for example in a biochemical pathway. Using knownmethods to investigate such interactions it would be necessary toidentify each gene involved in a pathway and conduct separatehybridisation reactions to determine the expression of each gene. Themethod of the invention, in contrast will in a single round ofhybridisation report on the comparative expression of all genesinvolved, even including genes that may not be known to be associatedwith the pathway.

Conveniently the method of the invention may be effected using an arrayor microarray on a solid substrate. Either the probe library or thereference samples may be provided as an array or microarray on asuitable substrate. Preferably it is the reference samples that areprovided as the array on the substrate. In a particularly preferredembodiment an array or microarray may comprise a library of referencesamples containing DNA samples derived from groups representative ofdifferent biological conditions, each group containing samples derivedfrom a number of different individuals sharing the same condition,wherein the DNA samples are arranged in order on the array or microarraysuch that members of the same group are located in proximity to oneanother. The DNA samples may, for instance, be arranged in order in agrid pattern such that each row of the grid represents a group ofindividuals sharing the same biological condition.

A suitable microarray may, for instance, be produced on a substrate suchas glass, a silica-based chip, a nylon membrane or a microtiter plate.Many examples of techniques suitable for the manufacture of arrays ormicroarrays will be readily apparent to those skilled in the art. Theseinclude the techniques disclosed in Maniatis et al. 1982, Chee et al.1996, Iyer et al. 1999, Lipshutz et al. 1995, Lockhart et al. 1995,Schena 1996, Schena et al. 1995, Soares et al. 1997 and Southern 1996.

DNA may be coupled to the support material forming the array by means ofelectrostatic interaction with a coating film of a polycationic polymersuch as poly-L-lysine (as described in WO 95/35505) or may be covalentlybound to the support by well established techniques.

In an alternative embodiment the method of the invention may, ifpreferred, be performed with both the probe library and referencesamples in free solution.

Biological samples suitable for use according to the invention includeany sample containing material representative of gene expression in thesample, such as mRNA. Biological samples preferably comprise biologicalcells, indeed a suitable biological sample may even comprise a singlecell. Suitable samples may be taken by means of biopsies, swabs, hair orskin samples, or as samples of bodily fluids such as blood,cerebrospinal fluid (CSF), saliva, milk, faeces and urine. In particularsamples for use in analysis of stem cells may suitably be taken fromfoetal or embryonic tissue, from bone marrow or from germ cells or fromother tissues in the adult or developing organism.

The probe library and the reference samples may preferably comprise, orbe derived from, global amplified cDNA, i.e. a cDNA population in whichall DNA sequences are present in the same relative abundance as the mRNAfrom which they have been derived. Most preferably the global amplifiedcDNA is prepared from mRNA using limiting concentrations of nucleotidesand a relatively short incubation time in order to limit cDNA synthesis.This ensures that, no matter what the length of the original mRNAtranscript, all cDNA molecules produced are of approximately the samerelatively small size. Since all the cDNA molecules are of approximatelyequal size subsequent amplification of the cDNA results in equalreproduction of all the cDNA molecules present. This ensures that theamplified cDNA produced reflects the original relative abundance of themRNA present in the biological sample. Suitable protocols for theproduction of global amplified cDNA of this nature are provided in Bradyet al. 1990, Cumano et al. 1992 and Brady et al. 1993. In addition tothe advantage of allowing the production of amplified populations ofcDNA that maintain the relative abundance of the original mRNA the useof global amplified cDNA also provides other advantages. For exampleglobal amplified cDNA can be derived either directly from one or morefreshly isolated living cells without the need for RNA isolation, orfrom mRNA purified from a biological sample. Additionally, theproduction of global cDNA is well suited to automation.

Materials that may be derived from total cDNA include sub-populations ofthe total cDNA, truncated or otherwise manipulated versions of the cDNAand other materials representative of patterns of gene expressionproduced using the total cDNA as a template.

The invention may be effected using probe libraries and/or referencesamples comprising cDNA produced as described above without furthermodification. However, various modifications may be made what willimprove the sensitivity of the method. For example, cDNA, or aderivative thereof, generated from the sample of interest or thebiological reference samples may be treated with a suitable 3′ or 5′exonuclease.

Thus treatment of the double stranded cDNA molecules generated from thesample of interest or reference samples with exonuclease results indegradation from either the 3′ or 5′ end of each strand of DNA(depending on the specificity of the enzyme selected). Thus regions ofeach strand that are complementary to one another are removed bydigestion. Digestion with double-stranded DNA (dsDNA) exonucleases willinitiate digestion at each end of a double-stranded DNA molecule. Sincethe dsDNA exonuclease preferentially removes one strand of thedouble-stranded molecule digestion tends to be “self-limiting”, and willdecrease when there are no remaining regions of double-stranded DNA.Thus the exonuclease treatment can effectively convert each startingdouble-stranded DNA molecule into two non-complementary single-strandedDNA molecules corresponding to the 3′ or 5′ “halves” of the originalmolecule.

Alternatively with knowledge of the average size of molecules within thedouble-stranded DNA population (determined, for example, by gelelectrophoresis) and the rate of digestion by the chosen exonuclease itis possible to chose an incubation period such that the digestionremoves a chosen length of the DNA molecules. This chosen length may,for example, be approximately half the average DNA molecule lengthpresent in the starting double-stranded DNA population. Such a digestwill, as with the technique described above, produce two single-strandedDNA molecules corresponding to the (3′ or 5′) “halves” of the lengths ofthe two starting strands of the original double-stranded DNA.

It will be appreciated that as a result of the digestion the tworemaining molecules are not complementary to one another. This thereforeprevents the strands of, for example, the probe library molecules (i.e.molecules derived by exonuclease digestion of cDNA from the sample ofinterest) re-hybridising to their complementary sequences found withinthe original double stranded DNA population from the sample of interest.Thus these DNA molecules are maintained in single stranded form withinthe probe library and are therefore free to hybridise to complementarysequences in the reference samples (should such sequences be present),thereby improving the sensitivity of the method of the invention. It ispreferred that both the library of probe molecules and the referencesamples are prepared using exonuclease digestion as described above.

In practice, in order to maximise sensitivity the specificity of theexonucleases will be reversed for probe and reference samples in orderto ensure that probe sequences will complement and therefor efficientlyhybridise to reference samples. For example, in one embodiment the probewill be treated with a 3′-5′ exonuclease and reference samples treatedwith a 5′-3′ exonuclease. In an alternative embodiment the probe will betreated with a 5′-3′ exonuclease and reference samples treated with a3′-5′ exonuclease.

Complexity reduction techniques may also be used in preparation of theprobe library and/or reference samples to improve the sensitivity of themethod of the invention. The rationale behind such techniques is thatmany of the mRNAs present in a biological sample, such as the sample ofinterest or the reference samples, represent transcription of so-called“house keeping” genes. These genes encode products associated with theup-keep of the cell and are generally likely to be common to bothsamples of interest and reference samples. As such they representcomponents of gene expression patterns that may be found in both testand reference samples, but which are unlikely to be important in thedevelopment or maintenance of a biological condition or state ofinterest. It has been estimated that up to 65% of mRNA mass within cellsmay be composed of transcripts representing “house-keeping” genes.

Complexity reduction techniques improve sensitivity either by simplyreducing the number of individual genes represented in the probe libraryand/or reference samples, or by specifically removing irrelevant or“house keeping” genes from the probe library and/or reference samples.Thus the relative abundance of those molecules representative of geneexpression that remain after application of a complexity reductiontechnique is increased, thereby increasing the “signal to noise” ratio.

A number of complexity reduction techniques may be used in effecting themethod of the invention. These techniques may be used in isolation or incombination. Preferably the same complexity reduction technique, orcombination of complexity reduction techniques, are used to treat thecDNA, or its derivatives, to produce both the probe library and thereference samples, although it is possible to apply complexity reductiontechniques to only one of the DNA populations.

Suitable Examples of Complexity Reduction Techniques Include:

Restriction Enzyme Based.

In this complexity reduction technique site specific endonucleases areused to digest the cDNA or its derivatives. Since the frequency ofcleavage sites for any specific endonuclease will depend on the size andbase composition of the cleavage site endonucleases can be chosen thatwill cut a sub-set of all DNA molecules present. For example, arestriction endonuclease recognising a six base site will, on average,cleave every 4,096 base pairs. Thus in a DNA population in which theaverage polynucleotide size is 2,000 bases such a endonuclease willcleave approximately half of all polynucleotides present. Followingrestriction digestion either the cleaved products or the uncleavedproducts can be selectively enriched. By choosing the appropriaterestriction enzymes distinct subsets of cDNA sequences can be eithereliminated or enriched. By applying this type of strategy the initialtotal cDNA sample can divided into subsets of genes whereby eachsequence is effectively enriched making it more likely that changes ineach individual gene will be detected during array hybridisation.

Thus for individual gene sequence present after applying complexityreduction there will be an increase in specific activity for each geneand an increase in the “signal to noise”.

Display Products.

Another means of selecting a subset of sequences present in the startingcDNA/mRNA population, and thereby increasing the relative abundance ofeach selected sequence after complexity reduction, is the use ofapproaches for differential cDNA display (Liang and Pardee, 1992). cDNAdisplay selectively amplifies only those cDNA populations which containeffective priming sites for display primer(s) used. Display primers canbe used to prepare distinct subsets of cDNAs directly from starting RNA(Liang and Pardee, 1992) or alternatively display amplification may beapplied to amplified total cDNA populations (Candeliere et al., 1999).In essence display techniques reduce complexity by selectively enrichinga subset of the sequences present in the original mRNA or cDNApopulation, thereby increasing the relative abundance of the selectedsequences within the resultant population.

Hybridisation Depletion and Enrichment.

A variety of DNA/RNA subtraction techniques have been developed todeplete DNA/RNA sequences common to two or more pools of DNA/RNAmolecules. DNA/RNA subtraction applied to DNA or RNA copies (eitherdirect copies or amplified products) of the original extracted RNA canbe used to reduce complexity by removing sequences.

Suitable DNA/RNA subtraction techniques for use according to theinvention are well known. One such method involves the production of asingle-stranded cDNA library (the “tracer”), such as the cDNA from whichthe probe library or reference samples are to be generated, from whichit is desired to remove certain sequences. A collection of amplifiedcDNAs representing the sequences that one wishes to eliminate (the“driver”), such as housekeeping genes, is then allowed to hybridise withthe tracer. Double stranded DNA molecules, representing hybrids of thetracer and the driver, may then be removed from the total population ofDNA based upon their adhesion to hydroxyapatite. The remaining DNApopulation comprises single stranded DNA molecules representing thetracer population minus the driver population. This subtracted DNApopulation may then be further amplified as required.

In further refinements of this method “driver” nucleic acids may becovalently linked to compounds which facilitate the physical separationof “driver” nucleic acids (plus any annealed “tracer”) from unhybridised“tracer”. The separated populations (i.e. those sequences present onlyin the “tracer”, or those sequences shared by both “tracer” and“driver”) may then be enriched or depleted relative to one another. Forexample, driver nucleic acids may be linked to biotin, such thatfollowing hybridization all biotinylated hybrids can by segregated byinteraction with immobilised avidin, allowing either subtractiveenrichment or positive selection. Suitable protocols are described inWelcher et al., 1986; and Weaver et al., 1999. In alternative, butsimilar, approaches “driver” nucleic acids may be bound to latex beads(as described in Kuribayashi-Ohta et al., 1993, or magnetic particles(as described in Lopez-Fernandez and del Mazo, 1993; and Schraml et al.1993.

In one embodiment hybridisation depletion/enrichment protocols can beused to remove “unwanted sequences” present in samples from which theprobe library and/or reference samples are derived. The nature of the“unwanted sequences” will depend on the biological samples in question.However, as a general rule, sequences which are expressed at similarlevels in diverse samples are, by their very nature, uninformative andtend simply to add to the “background” produced during hybridisation.

It is likely that genes expressed at a similar level in biologicallydivergent tissues will not be characteristic of a particular tissue, andwill instead represent house-keeping genes. By way of example, it isunlikely that genes expressed at a similar level in tissues asbiologically different as heart, lung, spleen and testes will becharacteristic of any one of these tissues. Sequential hybridisationenrichment can be used to obtain a “pool” of sequences common todifferent tissues. The resultant pool will represent genes thatcontribute to the “background noise” associated with hybridisation. Thispool can then be expanded and used to reduce the level of backgroundhybridisation. For example, it is possible to subtract these commonsequences from both the probe library and reference samples, therebyreducing the level of total hybridisation. Alternatively it is possibleto use the pool of common genes to produce unlabelled competitor DNA andthereby reduce the level of detectable hybridisation.

Using probe libraries and reference samples produced in accordance withthe techniques described above the method of the invention may beeffected by reference samples and probe library under hybridisingconditions. The conditions under which nucleic acids will hybridise toone another are well known to those skilled in the art. Specificconditions are described in greater detail in the accompanying Example.Further examples of conditions suitable for nucleic acid hybridisationcan be found in reference works such as “Molecular Cloning: A LaboratoryManual” edited by Maniatis et al. Other suitable conditions aredescribed in Chee et al. 1996, Iyer et al. 1999, Lipshutz et al. 1995,Lockhart et al. 1995, Schena 1996, Schena et al. 1995, Soares et al.1997 and Southern 1996.

Similarly, methods for determining the relative degree of hybridisationbetween populations of nucleic acids are also well known. Methodssuitable for effecting the invention include labelling of the probelibrary with reporters such as fluorescent labels, radioactive labels orchromogenic enzymes. If the reference sample libraries are unlabelledthen detection of the chosen label (after removing unbound probe) willconfirm the presence of hybridisation between the sample of interest andthe reference sample. Suitable techniques for labelling of the moleculescomprising the probe library, for detection of hybridised probe andreference DNA molecules and for interpretation of hybridisation data arewell known to those skilled in the art. These techniques include thosedescribed in Maniatis et al. 1982, Chee et al. 1996, Iyer et al. 1999,Lipshutz et al. 1995, Lockhart et al. 1995, Schena 1996, Schena et al.1995, Soares et al. 1997 and Southern 1996.

Use of Unlabelled Competitor DNA.

When the probe library DNA is labelled and the reference sample DNA isunlabelled the sensitivity of the method of the invention may beimproved by the use of unlabelled “competitor” DNA which can competewith the DNA of the probe library for hybridisation with the referencesamples. The competitor DNA may be DNA representing common housekeepinggenes, or it may be selected DNA representing genes common to thebiological sample of interest and/or the reference samples. Since thecompetitor DNA is unlabelled, hybrids of competitor and reference DNAwill not be detected in assessing total hybridisation.

The competitor DNA may be exposed to the reference sample DNA before theaddition of the probe library DNA or at the same time as the addition ofthe probe library DNA. Molecules of the competitor DNA that representgenes expressed by the reference samples will then hybridise to thecorresponding DNA of the reference samples. Reference sample moleculesthat undergo hybridisation with molecules of the competitor DNA willtherefore be unable to hybridise with further molecules from the probelibrary. Thus by incubating the DNA of the reference samples with, forexample, unlabelled competitor DNA representative of housekeeping genesit is possible to reduce the level of binding by labelled probe DNArepresenting the same genes. This therefore improves the sensitivity ofthe method of the invention since it increases the likelihood thatdetected hybridisation is representative of genes of interest within thesamples.

Unlabelled competitor DNA representative of genes having a highfrequency of expression within the biological sample of interest and/orreference samples may be generated by reverse subtraction of the DNApopulations derived from the two samples.

The present invention will now be illustrated by way of example onlywith reference to the accompanying drawing in which:

FIG. 1 a represents a schematic depiction of an array of referencesamples suitable for use in the method of the invention before effectinghybridisation;

FIG. 1 b represents the same array after effecting hybridisation of aprobe library with the reference samples;

FIG. 1 c represents a flow chart indicating suitable methods forproducing a probe library and reference samples according to theinvention; and

FIG. 1 a shows an array (1) provided with individual reference samples(2) derived from cDNA generated from biological reference samples. Eachindividual reference sample is a library representative of a pattern ofgene expression in the biological reference sample. The rows ofreference samples (2) on the array (1) each represent a distinctbiological condition or state. Each reference sample (2) within a row isderived from a different individual sharing the same biologicalcondition or state.

FIG. 1 b shows the results of probing the reference samples (2) on thearray (1) with a labelled probe library according to the method of theinvention. Sequences present within both the probe library and thereference samples (2) hybridise to one another. Hybridisation ismeasured by colour development, hence the greater the degree ofhybridisation between the probe library and a reference sample the moreintense the colour. Thus in FIG. 2 a it can be seen that the probelibrary exhibits the greatest degree of similarity (and sohybridisation) with the reference samples of row 10, a lesser degree ofsimilarity (and hybridisation) with reference samples of rows 3 and 6and a still lesser degree of similarity with the reference samples ofrows 1 and 8. The probe library does not share any sequences in commonwith the other rows of reference samples (2) and thus does not hybridisewith these reference samples, so producing no colour development.

The probe library and reference samples may be prepared by theprocedures illustrated in FIG. 1 c, in which RNA (3) from a biologicalsample of interest is amplified according to known protocols to generateglobal cDNA (4). This global cDNA (4) may then be used directly toproduce a probe library (as indicated by arrow 5) or, more preferably,is subjected to complexity reduction techniques (6) prior to probelibrary production.

Complexity reduction (6) may, for instance, take the form of processingto display products (7), subtraction of unwanted sequences from theglobal cDNA generated from the sample of interest (8) or restrictiondigest of sequences in the cDNA generated from the sample of interest(9). The cDNA generated from the sample of interest may be subject to acombination of complexity reduction techniques (e.g. subtraction (8) andrestriction digest (9)) or may be used to produce a probe library aftera single complexity reduction technique.

Optionally, the cDNA, or derivative, of the probe library may be subjectto exonuclease digestion in order to improve the sensitivity of theinvention. This digestion may be effected either before or aftercomplexity reduction.

Production of the probe library is completed by using known techniquesto label (10) the cDNA generated from the sample of interest.

Although the generation and processing of cDNA has been described abovewith reference to production of the probe library, the techniquesdescribed (with the exception of labelling the probe library) are allequally suitable for production of reference samples from biologicalreference samples in order to produce a suitable array (11). Preferablyboth the probe library and reference samples to be used according to themethod of the invention are produced using the same complexity reductiontechniques. In the situation that both the probe library and referencesamples are to be subject to exonuclease digestion the two differentcDNA populations should be treated with exonucleases having differentspecificities, i.e. one treated with a 5′ to 3′ exonuclease, and theother treated with a 3′ to 5′ exonuclease.

Protocols.

The following Protocols are suitable for effecting the method describedabove using a collection of labelled target DNA molecules according tothe invention as a “probe library”.

-   -   (a) Preparation of global amplified cDNA        -   (i) Preparation of cDNA        -   (ii) Terminal transferase—“Tailing”        -   (iii) Global cDNA amplification    -   (b) Preparation of array of reference samples    -   (c) Labelling of probe library        -   (i) Terminal Transferase labelling        -   (ii) PCR labelling    -   (d) Exonuclease treatment of double-stranded DNA    -   (e) Hybridisation of probe library and reference samples    -   (f) Detection of hybridisation    -   (g) Complexity reduction.        -   (i) Display Based        -   (ii) Hybridisation depletion and enrichment            (a) Preparation of Global Amplified cDNA.

The protocol described below is based on protocols described in Brady etal. (1990) and Brady, G., and Iscove, N. N. (1993).

Suitable starting materials include total RNAs, which may be preparedfrom biological tissues of interest (using commercially available kitssuch as those manufactured by Clontech), or mRNA present in biologicalcells (“direct amplification”).

(i) Preparation of cDNA.

cDNA may be prepared from the mRNA from the biological tissues accordingto the following protocol:

1. RNAs are adjusted to 100 microgram/ml in 10 mM Tris pH 7.5, 1 mM EDTA

2. 3 μl of each RNA is added to 3 μl of the following buffer: 100 mMTris pH 8.3 150 mM KCl 6 mM MgCl₂ 0.2 mg/ml Glycogen (Roche) 2% NP-40(Roche) 2.5 nM dNTPs (Sigma) 0.75 μM dT24 (Sigma/Genosys) 0.37 u/mlRNAse inhibitors (Ambion)

3. Samples are heated to 65° C. for 1 minute allowed to cool at RT for 3minutes then placed on wet ice

4. After 1 to 10 minutes on ice 3 μl of the following buffer containing85 u MMLV RTase and 1 u AMV RTase is added to each sample: 50 mM Tris pH8.3 75 mM KCl 3 mM MgCl₂ 0.1 mg/ml Glycogen (Roche) 1% NP-40 (Roche)

5. Samples are Incubated 15 minutes at 37° C., heat inactivated at 65°C. for 10 minutes then cooled to 4° C.

(ii) Terminal Transferase—‘Tailing’

1. 5 μl of each sample is mixed with 5 μl of the following buffercontaining 2.3 units terminal transferase. 200 mM potassium cacodylatepH 7.2 4 mM CoCl₂ 0.4 mM DTT 1 mM dATP

2. Samples are then incubated 15 minutes at 37° C., 65° C. 10 minutesand cooled to 4° C.

(iii) Global cDNA Amplification.

Global cDNA prepared from biological tissues according to the precedingprotocols may be amplified according to the following protocol:

1. 8 μl of the tailed cDNA prepared as described above may be combinedwith 8 μl of: 121.4 mM KCl   8.5 mM MgCl₂ 24.25 mM Tris-HCl pH 8.3 48μg/ml Glycogen (Roche) 2.4% Triton X-100   2.3 mM dNTPs 9.6 μMOligo           Not1dT              (sequenceCATCTCGAGCGGCCGCTTTTTTTTTTTTTTTTTTTTTTTT) 0.16 u/μl Taq Polymerase

2. Samples are then placed into a PCR machine and subjected to:

-   25 cycles-   1 minute 94° C.-   2 minute 42° C.-   6 minute 72° C.    followed by an additional 25 cycles:-   1 minute 94° C.-   1 minute 42° C.-   2 minute 72° C.

3. Following completion of PCR samples are purified using the Millipore96 well purification system (Millipore MANU 03050) followinginstructions provided by the manufacturer.

(b) Preparation of Array of Reference Samples.

An array comprising global purified cDNA (prepared as described above)may be produced using the following protocol:

Purified global cDNAs from heart, lung, spleen and testes may separatelybe adjusted to around 50 ng/μl in 50% DMSO, boiled and spotted in groupsof 12 onto CMTGAPS glass slides (Corning) using a Gene Machines OmniGridas recommended by the manufacturer.

(c) Labelling of Probe Library.

The following provides suitable protocols for labelling of probe librarycDNA for use according to the method of the invention. The followingprotocols describes the labelling of two different cDNA populations(which may be prepared using the protocols described above) with twodifferent fluorescent markers (Cy3 and Cy5).

(i) Terminal Transferase Labelling.

1. Approximately 50 ng of globally amplified cDNA of a first probelibrary may be added to a 20 μl reaction containing: 100 nM FluoroLink ™Cy3-dUTP (Amersham Pharmacia Biotech) 100 mM potassium cacodylate pH 7.22 mM CoCl₂ 0.2 mM DTT total 5 units Terminal Transferase

2. Approximately 50 ng of globally amplified cDNA of a second probelibrary may be added to a 20 μl reaction containing: 100 nM FluoroLink ™Cy5-dUTP (Amersham Pharmacia Biotech) 100 mM potassium cacodylate pH 7.22 mM CoCl₂ 0.2 mM DTT total 5 units Terminal Transferase

3. Following incubation for 1 hour at 37° C. both samples may be ethanolprecipitated by the addition of: 10 μl 7.5 M Ammonium Acetate 0.5 μl 15mg/ml Glyco Blue (Ambion) 75 μl ethanol

Samples may then be held on wet ice for 15 minutes, centrifuged at 4° C.at 14,000 rpm for 20 minutes and the pellets washed twice with 70%ethanol, allowed to dry 10 minutes at room temperature then resuspendedin 5 μl 10 mM Hepes pH 7.5, 1 mM EDTA.

(ii) PCR Labelling.

Further rounds of PCR amplification can be used to incorporatefluorescent markers directly or indirectly coupled to nucleotidespresent in the PCR reaction. An example of such an approach is givenbelow.

1. Approximately 0.5 ng of globally amplified cDNA of a first probelibrary may be added to a 20-100 μl reaction containing: 100 nMFluoroLink ™ Cy3-dUTP (Amersham Pharmacia Biotech) 100 nM dNTPs 1 μMOligo           Not1dT              (sequenceCATCTCGAGCGGCCGCTTTTTTTTTTTTTTTTTTTTTTTT)  16 mM (NH₄)₂SO₄  67 mMTris-HCl (pH 8.8 at 25° C.) 0.01% Tween-20 0.16 u/μl Taq Polymerase

2. Approximately 0.5 ng of globally amplified cDNA of a second probelibrary may be added to a 20-100 μl reaction containing: 100 nMFluoroLink ™ Cy5-dUTP (Amersham Pharmacia Biotech) 100 nM dNTPs 1 μMOligo           Not1dT              (sequenceCATCTCGAGCGGCCGCTTTTTTTTTTTTTTTTTTTTTTTT)  16 mM (NH₄)₂SO₄  67 mMTris-HCl (pH 8.8 at 25° C.) 1.5 mM MgCl₂ 0.01% Tween-20 0.16 u/μl TaqPolymerase

3. Both samples are then placed into a PCR machine and subjected to: 25cycles 30 seconds 94° C. 1 minute 42° C. 2 minutes 72° C.

4. Following both samples may be ethanol precipitated by the additionof: 0.5 original sample volume 7.5 M Ammonium Acetate 0.025 originalsample volume 15 mg/ml Glyco Blue (Ambion) 3.5 original sample volumesethanol

Samples may then be held on wet ice for 15minutes, centrifuged at 4° C.at 14,000 rpm for 20 minutes and the pellets washed twice with 70%ethanol, allowed to dry 10 minutes at room temperature then resuspendedin 5 μl 10 mM Hepes pH 7.5, 1 mM EDTA.

(d) Exonuclease Treatment of Double-Stranded DNA.

Note exonuclease treatment can be applied to either freshly amplifiedcDNA or labelled cDNA.

(i) 3′-5′-Exonuclease—Exonuclease III

1. Add freshly 0.5-5 μg cDNA to a 50 μl reaction consisting of: 660 mMTris pH 8.0 6.6 mM MgCl₂ 5 mM DTT 50 μg/ml BSA 10 units Exonuclease III(Amersham Pharmacia)

2. Incubate 30 minutes at 37° C.

3. Following heat inactivation at 75° C. for 30 minutes ethanolprecipitate by the addition of: 0.5 original sample volume 7.5 MAmmonium Acetate 0.025 original sample volume 15 mg/ml Glyco Blue(Ambion) 3.5 original sample volumes ethanol

Samples may then be held on wet ice for 15 minutes, centrifuged at 4° C.at 14,000 rpm for 20 minutes and the pellets washed twice with 70%ethanol, allowed to dry 10 minutes at room temperature then resuspendedin 5 μl 10 mM Hepes pH 7.5, 1 mM EDTA.

(ii) 5′-3′-Exonuclease—T7 Gene 6 Exonuclease

1. Add freshly 0.5-5 μg cDNA to a 50 μl reaction consisting of: 40 mMTris pH7.5 20 mM MgCl₂ 50 mM NaCl 10 units T7 Gene 6 Exonuclease(Amersham Pharmacia)

3. Incubate 30 minutes at 37° C.

3. Following heat inactivation at 75° C. for 30 minutes ethanolprecipitate by the addition of:  0.5 original sample volume 7.5 MAmmonium Acetate 0.025 original sample volume 15 mg/ml Glyco Blue(Ambion)  3.5 original sample volumes ethanol

Samples may then be held on wet ice for 15 minutes, centrifuged at 4° C.at 14,000 rpm for 20 minutes and the pellets washed twice with 70%ethanol, allowed to dry 10 minutes at room temperature then resuspendedin 5 μl 10 mM Hepes pH 7.5, 1 mM EDTA.

(e) Hybridisation of Probe Library and Reference Samples.

Hybridisation of probe library and reference samples according to themethod of the invention may be effected as follows, using an array andprobe libraries prepared as described above.

1. An array slide may be prehybridised at 42° C. for 1 hour in thefollowing buffer:

-   50% Formamide-   5×SSC-   0.1% SDS-   10 mg/ml BSA

2. The array slide may then be washed four times with H₂O and once inIsopropanol and dried 5 minutes at room temperature.

3. The following mixture may then be prepared: 50% v/v Formamide 5X SSC0.1% SDS 0.5 mg/ml Poly A RNA 0.5 mg/ml Yeast tRNA 0.5 mg/ml SalmonSperm DNA (10-30 ug)  50 ug/ml Cot1 DNAcombined Cy3 and Cy5 probes(Total volume 45 μl)

4. The mixture may then be heated at 95° C. for 5 minutes and chilled onwet ice 3 minutes.

5. The mixture may be applied to a cover slip and the pre-warmed (42°C.) array slide (arrayed material facing downwards) lowered onto coverslip to the point when it is just possible to lift the cover slip upwith surface tension.

6. The slide may be placed into a moisturised slide hybridisationchamber and incubated 42° C. o/n.(<16 hr).

7. Following hybridisation the entire slide may be immersed in 2×SSC andthe cover slip removed.

8. The exposed slide may then be washed twice 2×SSC/0.1% SDS (5 minutesRT each wash) followed by 2 washes with 2×SSC (5 minutes RT each wash)and drying at room temperature.

(f) Detection of Hybridisation.

The following protocol is suitable for detection and analysis ofhybridisation in the method of the invention.

1. Scanning of the slide and quantification of red (Cy5 635 nm) andgreen (Cy3 532 nm) fluorescence may be carried out using a GenePix 4000bas recommended by the manufacturer.

2. Following scanning data may be analysed using commercially availablesoftware.

(g) Complexity Reduction.

There are many possible complexity reduction techniques that aresuitable for use with the method of the invention.

(i) Display Based

The following protocol is suitable for effecting a “display products”complexity reduction technique according to the method of the invention.The protocol provides for the preparation of two different amplifiedcDNA populations from the same cDNA population (“total cDNA”).

Selected subsets of cDNA within a global amplified total cDNA populationmay be further amplified based on protocols described in:

Candeliere, G. A., Rao, Y., Floh, A., Sandler, S. D., and Aubin, J. E.(1999). cDNA fingerprinting of osteoprogenitor cells to isolatedifferentiation stage-specific genes. Nucleic Acids Research 27,1079-83.

A suitable protocol is as follows:

1. Purified globally amplified total cDNA prepared as described abovemay be diluted 100 fold in 2 mM Tris pH 7.5, 0.2 mM EDTA.

2. Two separate subsets of cDNAs may then be selectively amplified fromthe total cDNA by separately adding 10 μl of total cDNA to 10 μl of PCRmixture A and 10 1μl of total cDNA to 10 μl of PCR mixture B andsubjecting both to:

-   2 cycles as follows:-   94° C. 1 minutes;-   35° C. 3 minutes;-   72° C. 3 minutes;    followed by 30 cycles as follows:-   94° C. 30 seconds;-   50° C. 30 seconds;-   72° C. 1 minute; and    1 cycle as follows:-   72° C. 5 minutes.

PCR Mixture A 25 μM Display Oligo A - CAGCCAGTCTTGAGGCAACACC 0.5 mMdNTPs (Sigma)  32 mM (NH₄)₂SO₄ 134 mM Tris-HCl (pH 8.8 at 25° C.) 0.01%Tween-20   3 mM MgCl₂ 25 u/ml Taq Polymerase

PCR Mixture B 25 μM Display Oligo B - CCAGCAAGAGCACAAGAGGAAGAG 0.5 mMdNTPs (Sigma)  32 mM (NH₄)₂SO₄ 134 mM Tris-HCl (pH 8.8 at 25° C.) 0.01%Tween-20   3 mM MgCl₂ 25 u/ml Taq Polymerase

Following PCR all samples may be purified using GFX purification columns(Amersham Pharmacia) following the manufacturer's instructions.

(ii) Hybridisation Depletion and Enrichment

The term driver refers to the cDNA used to deplete and/or enrich in thetracer cDNA population. The resultant depleted or enriched sequenceswill be derived from the tracer cDNA population. In the followingexamples all driver cDNAs are prepared in PCR reactions containing dUTP(not dTTP) to allow removal of residual driver cDNAs using the dUTPspecific UNG nuclease.

Based on Methods Described in:

Analysis of gene-expression in a complex differentiation hierarchy byglobal amplification of cdna from single cells. Brady, G, Billia F, KnoxJ, Hoang T, Kirsch I R, Voura E B, Hawley R G, Cumming R, Buchwald M,Siminovitch K, Miyamoto N, Boehmelt G, and Iscove N N: Current Biology1995, 5: 909-922.

Foot, H C C, Brady G, and Franklin F C H. (1996). SubtractiveHybridisation. In Plant Molecular Biology Laboratory Manual, M. Clark,ed. (London: Springer Verlag).

Weaver, D L, Núñez C, Brunet C, Bostock V, and Brady G. (1999).Single-cell RT-PCR cDNA subtraction. In Molecular Embryology: Methodsand Protocols., P. Sharpe and I. Mason, eds. (Totowa, N.J., USA: HumanaPress), pp. 601-609.

Depletion/Subtraction

1. Preparation of Tracer and Driver:

Tracer

Approximately 0.5 ng of globally amplified cDNA added to a 20-100 μlreaction containing: 250 nM dATP, dTTP, dCTP, dGTP 1 μMOligo           Not1dT              (sequenceCATCTCGAGCGGCCGCTTTTTTTTTTTTTTTTTTTTTTTT)  16 mM (NH₄)₂SO₄  67 mMTris-HCl (pH 8.8 at 25° C.) 1.5 mM MgCl₂ 0.01% Tween-20 0.16 u/μl TaqPolymeraseDriver

Approximately 0.5 ng of globally amplified cDNA added to a 20-100 μlreaction containing: 250 nM dATP, dUTP, dCTP, dGTP 1 μMOligo           Not1dT              (sequenceCATCTCGAGCGGCCGCTTTTTTTTTTTTTTTTTTTTTTTT)  16 mM (NH₄)₂SO₄  67 mMTris-HCl (pH 8.8 at 25° C.) 1.5 mM MgCl₂ 0.01% Tween-20 0.16 u/μl TaqPolymerase

Both tracer and driver are then placed into a PCR machine and subjectedto: 25 cycles 30 seconds 94° C.  1 minute 42° C.  2 minutes 72° C.

Following completion of the PCR reaction both tracer and driver cDNAsare then purified using commercial purification systems such as GFX(Amersham Pharmacia).

Biotinylation of Driver.

Place 20-50 μl driver DNA (2-50 μg) in a 1.5 ml screw-cap tube. Boil for2 minutes and place directly on ice in a small ice tray+rack.

Add 20 μl 2 mg/ml photobiotin to the DNA and mix well. With the lidsleft off place the tubes upright on ice 10 cm from the bulb andirradiate for a total 10 minutes. After the first 5 minutes remove thetubes from under the light source (avoid direct irradiation), flick thetube to mix and replace under the light source for the remaining 5minutes.

Remove the sample (avoid direct irradiation) and mix in the remaining 20μl of photobiotin and place under the light for an additional 5 minutes.

Add 1/10th volume of 1M Tris-Cl, pH 8.0 to stop the reaction.

Purify using commercial purification systems such as GFX (AmershamPharmacia).

2. Hybridisation of tracer plus driver and tracer enrichment:

To a 0.5 ml tube add and mix in this order: 0.5 μg tracer DNA  10 μgbiotinylated driver DNA

adjust volume to 20 μl with water then add:  8 μl 5xHyb GEH 12 μl 40%PEGHeat Sample:

-   5 minutes 98° C.,-   5 minutes at 80° C.-   7 minutes at 74° C.-   60 minutes at 68° C.-   then hold at 68° C. while separating biotinylated molecules

Remove biotinylated molecules using avidin bound to a solid support. Inpractise this can be carried out using commercial products as ditrectedby the manufacturer such as Streptavidin Magnasphere™ Paramagneticparticles (SA-PMPs) provided by Promega.

Following removal of biotinylated molecules the remaining tracer can besubjected to further rounds of subtraction by addition of freshbiotinylated driver DNA and repeating the process described above.Typically three sequential rounds of subtraction are used but additionalrounds may be added if required.

The final depleted product is then amplified using PCR conditionsdescribed for the original tracer amplification.

5×Hyb GEH   90 mM EPPS pH 8.5   10 mM EDTA pH 8.0 0.5% Triton X-100 3.75M NaClNegative Subtraction or Attraction1. Preparation of Tracer and Driver:Tracer

Approximately 0.5 ng of globally amplified cDNA added to a 20-100 μlreaction containing: 250 nM dATP, dTTP, dCTP, dGTP 1 μMOligo           Not1dT              (sequenceCATCTCGAGCGGCCGCTTTTTTTTTTTTTTTTTTTTTTTT)  16 mM (NH₄)₂SO₄  67 mMTris-HCl (pH 8.8 at 25° C.) 1.5 mM MgCl₂ 0.01% Tween-20 0.16 u/μl TaqPolymeraseDriver

Approximately 0.5 ng of globally amplified cDNA added to a 20-100 μlreaction containing: 250 nM dATP, dUTP, dCTP, dGTP 1 μMOligo           Not1dT              (sequenceCATCTCGAGCGGCCGCTTTTTTTTTTTTTTTTTTTTTTTT)  16 mM (NH₄)₂SO₄  67 mMTris-HCl (pH 8.8 at 25° C.) 1.5 mM MgCl₂ 0.01% Tween-20 0.16 u/μl TaqPolymerase

Both tracer and driver are then placed into a PCR machine and subjectedto: 25 cycles 30 seconds 94° C.  1 minute 42° C.  2 minutes 72° C.

Following completion of the PCR reaction both tracer and driver cDNAsare then purified using commercial purification systems such as GFX(Amersham Pharmacia).

Biotinylation of Driver

Place 20-50 μl driver DNA (2-50 μg) in a 1.5 ml screw-cap tube. Boil for2 minutes and place directly on ice in a small ice tray+rack.

Add 20 μl 2 mg/ml photobiotin to the DNA and mix well. With the lidsleft off place the tubes upright on ice 10 cm from the bulb andirradiate for a total 10 minutes. After the first 5 minutes remove thetubes from under the light source (avoid direct irradiation), flick thetube to mix and replace under the light source for the remaining 5minutes.

Remove the sample (avoid direct irradiation) and mix in the remaining 20μl of photobiotin and place under the light for an additional 5 minutes.

Add 1/10th volume of IM Tris-Cl, pH 8.0 to stop the reaction.

Purify using commercial purification systems such as GFX (AmershamPharmacia).

2. Hybridisation of tracer plus driver and tracer enrichment:

To a 0.5 ml tube add and mix in this order: 0.5-10 μg tracer DNA 10 μgbiotinylated driver DNA 1

adjust volume to 20 μl with water then add:  8 μl 5xHyb GEH 12 μl 40%PEG

Heat Sample:

-   5 minutes 98° C.,-   5 minutes at 80° C.-   7 minutes at 74° C.-   60 minutes at 68° C.-   then hold at 68° C. while separating biotinylated molecules

Remove biotinylated molecules using avidin bound to a solid support. Inpractise this can be carried out using commercial products as ditrectedby the manufacturer such as Streptavidin Magnasphere™ Paramagneticparticles (SA-PMPs) provided by Promega.

Release tracer DNA bound to driver DNA 1 by denaturing the driver DNA1/tracer DNA hybrids. For examples using SA-PMPs the washed SA-PMPs andtheir attendant driver DNA1/tracer DNA hybrids can be heated to 96° C.to release tracer DNA and bound driver DNA 1 removed by magneticattraction of the SA-PMPs.

Released tracer DNA can then be subjected to further rounds ofattraction by repeating the process with separate drivers (driver DNAs2, 3, 4 etc).

The final “attracted” product will be enriched for sequences common toall driver DNAs used and can be amplified using PCR conditions describedfor the original tracer amplification.

5×Hyb GEH 90 mM EPPS pH 8.5 10 mM EDTA pH 8.0 0.5% Triton X-100 3.75 MNaCl

REFERENCES

-   Brady, G., Barbara, M., and Iscove, N. N. (1990). Representative in    vitro cDNA amplification from individual hemopoietic cells and    colonies. Meth. Mol. Cell. Biol. 2, 17-25.-   Brady, G., and Iscove, N. N. (1993). Construction of cDNA libraries    from single cells. Methods Enzymol. 225, 611-623.-   Chee, M., Yang, R., Hubbell, E., Bemo, A., Huang, X. C., Stem, D.,    Winkler, J., Lockhart, D. J., Morris, M. S., and Fodor, S. P.    (1996). Accessing genetic information with high-density DNA arrays.    Science 274, 610-4.-   Cumano, A., Paige, D. J., Iscove, N. N. and Brady, G. (1992)    Bipotential precursors of B cells and macrophages in murine fetal    liver. Nature, 356, 612-615.-   Iyer, V. R., Eisen, M. B., Ross, D. T., Schuler, G., Moore, T.,    Lee, J. C., Trent, J. M., Staudt, L. M., Hudson, J., Jr.,    Boguski, M. S., Lashkari, D., Shalon, D., Botstein, D., and    Brown, P. O. (1999). The transcriptional program in the response of    human fibroblasts to serum. Science 283, 83-7-   Kuribayashi-Ohta, K, Tamatsukuri S, Hikata M, Miyamoto C, and    Furuichi Y “Application of oligo(dT)30-latex for rapid purification    of poly(A)+ mRNA and for hybrid subtraction with the in situ reverse    transcribed cDNA.”: Biochim Biophys Acta 1993, 1156: 204-12)-   Liang, P., and Pardee, A. B. (1992). Differential display of    eukaryotic messenger RNA by means of the polymerase chain reaction.    Science 257, 967-71.-   Lipshutz, R. J., Morris, D., Chee, M., Hubbell, E., Kozal, M. J.,    Shah, N., Shen, N., Yang, R., and Fodor, S. P. (1995). Using    oligonucleotide probe arrays to access genetic diversity.    Biotechniques 19, 442-7.-   Lockhart, D. J., Dong, H., Byrne, M. C., Follettie, M. T., Gallo, M.    V., Chee, M. S., Mittmann, M., Wang, C., Kobayashi, M., Horton, H.,    and Brown, E. L. (1996). Expression monitoring by hybridization to    high-density oligonucleotide arrays. Nat Biotechnol 14, 1675-80.-   Lopez-Fernandez, L A, and del Mazo J “Construction of subtractive    cDNA libraries from limited amounts of mRNA and multiple cycles of    subtraction.”: Biotechniques 1993, 15: 654-6, 658-9.-   Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982). Molecular    Cloning: A Laboratory Manual (Cold Spring Harbor, N.Y.: Cold Spring    Harbor University Press).-   Schena, M. (1996). Genome analysis with gene expression microarrays.    Bioessays 18, 427-31.-   Schena, M., Shalon, D., Davis, R. W., and Brown, P. O. (1995).    Quantitative monitoring of gene expression patterns with a    complementary DNA microarray. Science 270, 467-70.-   Schraml, P, Shipman R, Stulz P, and Ludwig CU “cDNA subtraction    library construction using a magnet-assisted subtraction technique    (MAST).”: Trends Genet 1993, 9: 70-1.-   Soares, M. B. (1997). Identification and cloning of differentially    expressed genes. Curr Opin Biotechnol 8, 542-6.-   Southern, E. M. (1996). DNA chips: analysing sequence by    hybridization to oligonucleotides on a large scale. Trends Genet 12,    110-5.-   Weaver, D L, Nufiez C, Brunet C, Bostock V, and Brady G. (1999).    “Single-cell RT-PCR cDNA subtraction. In Molecular Embryology:    Methods and Protocols.”, P. Sharpe and I. Mason, eds. (Totowa, N.J.,    USA: Humana Press), pp. 601-609).-   Welcher, A A, Torres A R, and Ward D C “Selective enrichment of    specific DNA, cDNA and RNA sequences using biotinylated probes,    avidin and copper-chelate agarose.”: Nucleic Acids Res 1986,14:    10027-44;

1-21. (canceled)
 22. A method of analysing a biological sample ofinterest, comprising: (i) providing a probe library which comprises cDNAor a derivative thereof representative of a pattern of multiple geneexpression in the biological sample of interest; (ii) providing aplurality of individual reference samples each being a library comprisedof cDNA or a derivative thereof representative of a pattern of geneexpression in reference biological samples from which the referencesamples have been derived; (iii) treating individual reference sampleswith the probe library under hybridising conditions; and (iv)determining the relative degree of hybridisation of the probe library tothe reference samples, thereby providing an indication of the degree ofsimilarity between gene expression in the biological sample of interestand gene expression in the individual reference biological samples. 23.A method according to claim 22, wherein the reference samples areprovided as an array on a substrate.
 24. A method according to claim 22,wherein the reference samples comprise cDNA or a derivative thereofderived from biological reference samples representing a number ofdifferent biological conditions or states.
 25. A method according toclaim 22, wherein the reference samples comprise cDNA or a derivativethereof derived from biological reference samples representing a numberof different examples of the same biological condition or state.
 26. Amethod according to claim 22, wherein the probe library is prepared by acomplexity reduction technique from cDNA obtained from the biologicalsample of interest.
 27. A method according to claim 22, wherein thereference samples are prepared by a complexity reduction technique fromcDNA obtained from the reference biological samples.
 28. A method asclaimed in claim 26, wherein the complexity reduction techniquecomprises a restriction digestion technique.
 29. A method as claimed inclaim 26, wherein the complexity reduction technique comprises asubtraction technique.
 30. A method as claimed in claim 26, wherein thecomplexity reduction technique comprises a cDNA display technique.
 31. Amethod as claimed in claim 22, wherein the hybridisation is effected inthe presence of competitor DNA.
 32. A method according to claim 22,wherein the probe library is labelled with a fluorophore in order todetermine the relative degree of hybridisation of the probe library tothe reference samples.
 33. A method according to claim 22, wherein theprobe library or reference samples are subject to partial exonucleasedigestion prior to effecting hybridisation.
 34. A method according toclaim 33, wherein both the probe library and the reference samples aresubject to partial exonuclease digestion prior to effectinghybridisation, and the probe library and reference samples are treatedwith exonucleases having different specificities.
 35. A method accordingto claim 22, wherein the probe library and/or reference samples comprisea cDNA derivative and said derivative is RNA.
 36. A collection ofindividual reference samples each being a library comprised of cDNA or aderivative thereof representative of a pattern of gene expression inreference biological samples from which the reference samples have beenderived.
 37. A collection of individual reference samples as claimed inclaim 36, wherein the reference samples comprise cDNA or a derivativethereof derived from biological reference samples representing a numberof different examples of the same biological condition or state.
 38. Acollection of individual reference samples as claimed in claim 36,wherein the reference samples are prepared by a complexity reductiontechnique from cDNA obtained from reference biological samples.
 39. Acollection of individual reference samples as claimed in claim 38,wherein the complexity reduction technique comprises a restrictiondigestion technique.
 40. A collection of individual reference samples asclaimed in claim 38, wherein the complexity reduction techniquecomprises a subtraction technique.
 41. A collection of individualreference samples as claimed in claim 38, wherein the complexityreduction technique comprises a cDNA display technique.
 42. An array ormicroarray that comprises a collection of reference samples as claimedin claim 36.